JordanA. Shavit,1Ani Manichaikul,2Heidi L. Lemmerhirt,3Karl W. Broman,2and David Ginsburg1,3,4
1Department of Pediatrics, University of Michigan,AnnArbor;2Department of Biostatistics, Johns Hopkins University, Baltimore, MD;3Department of Human
Genetics, and4Howard Hughes Medical Institute and Department of Internal Medicine, University of Michigan,AnnArbor
Type 1 von Willebrand disease (VWD) is
the most common inherited human bleed-
ing disorder. However, diagnosis is com-
plicated by incomplete penetrance and
variable expressivity, as well as wide
variation in von Willebrand factor (VWF)
levels among the normal population. Pre-
vious work has exploited the highly vari-
able plasma VWF levels among inbred
strains of mice to identify 2 major regula-
tors, Mvwf1 and Mvwf2 (modifier of VWF).
is a natural variant in Vwf that alters
biosynthesis. We report the identification
of an additional alteration at the Vwf lo-
cus (Mvwf5), as well as 2 loci unlinked to
Vwf (Mvwf6-7) using a backcross ap-
proach with the inbred mouse strains
WSB/EiJ and C57BL/6J. Through posi-
tional cloning, we show that Mvwf5 is a
cis-regulatory variant that alters Vwf
mRNA expression. A similar mechanism
centage of human VWD cases, especially
those with no detectable mutation in the
VWF coding sequence. Mvwf6 displays
conservation of synteny with potential
VWF modifier loci identified in human
pedigrees, suggesting that its ortholog
may modify VWF in human populations.
von Willebrand factor (VWF) is a central component of hemosta-
sis, serving as the adhesive link between platelets and the injured
blood vessel wall, as well as the carrier for factor VIII. Deficiencies
in VWF result in von Willebrand disease (VWD), the most
common inherited bleeding disorder in humans. Elevated VWF
levels may also be an important risk factor for thrombosis, both
through a direct role in platelet adhesion,1as well as indirectly by
causing elevated levels of factor VIII.2-4Diagnosis of VWD is
elusive in many cases because of its variable expressivity and
incomplete penetrance5and the nonspecific nature of bleeding
symptoms.6VWF plasma protein levels also display a broad
distribution in the normal human population. Thus, it is often
difficult to determine whether a person has VWD and is at risk for
pathologic hemorrhage or simply has VWF levels in the low range
Levels of plasma VWF have been shown to be largely
determined by genetic factors, with estimates of heritability in
humans ranging from 25% to 32% by pedigree analysis,7,8to 66%
to 75% in twin studies.9,10ABO blood group is responsible for
one-third of the genetic variability in VWF plasma levels.11
However, the loci responsible for the remaining two-thirds of this
genetic component are unknown. Recent evidence from European
and Canadian cooperative studies on type 1 VWD have found that
disease diagnosis does not segregate with VWF genotype in
approximately 50% of families, supporting the existence of addi-
tional genetic factors.12-16
Laboratory mice display wide variation in VWF levels with
strikingly similar to the estimates for humans derived from twin
studies.9,10This variability among inbred mouse strains has been
used to identify genetic loci modifying VWF levels, including
Mvwf1 (modifier of Vwf), a mouse glycosyltransferase (B4galnt2)
that alters clearance of VWF.18A similar mechanism probably
explains the modification of human VWF levels by ABO blood
group and some cases of type 1 VWD.19,20A natural variant of the
murine Vwf gene has also been identified (Mvwf2).17
We now report a backcross between 2 additional inbred mouse
strains, WSB/EiJ (WSB) and C57BL/6J (B6), with relatively high
and low levels of plasma VWF, respectively. Genetic analysis
identified 3 significant loci regulating VWF levels. The first is a
novel cis-regulatory allele of Vwf, whereas the others map to novel
loci on chromosomes 5 and 10.
Mouse strains and bleeding
Mice were purchased from The Jackson Laboratory. Each individual mouse
was bled on 3 separate occasions, at least 1 week apart. Bleeds were
performed after isoflurane-induced anesthesia by retro-orbital technique on
alternating eyes from week to week, removing approximately 75 ?L whole
blood with each bleed into heparinized capillary tubes (Thermo Fisher
Scientific). For the strain survey, 3 females of each strain were bled between
4 and 8 weeks of age. Further analysis of the C57BL/6J (B6) and WSB/EiJ
(WSB) strains were as follows: 2 each of WSB males and females, 3 males
and 4 females of B6, and 4 males and 5 females of (B6 ? WSB) F1 mice
were bled between 3 and 8 weeks of age. For the backcross study, WSB
males were crossed to B6 females to generate F1 progeny. Both male and
female F1s were backcrossed to B6 to produce the N2 generation.
Two hundred seven N2 mice were also bled in the same manner described
above, with the first bleed at weaning, the second between 3.5 and
Submitted July 15, 2009; accepted August 15, 2009. Prepublished online as
Blood First Edition paper, September 29, 2009; DOI 10.1182/ blood-2009-07-
An Inside Blood analysis of this article appears at the front of this issue.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2009 by TheAmerican Society of Hematology
5368 BLOOD, 17 DECEMBER 2009?VOLUME 114, NUMBER 26
6.5 weeks, and the third from 5 to 8.5 weeks. The mice were then
exsanguinated by cardiac puncture after pentobarbital-induced anesthesia,
and blood was collected into 0.5M EDTA pH 8.0 at a dilution of 1:40.
Organs were harvested for genomic DNApreparation. Mice were housed in
microisolator cages, and all procedures were approved by and performed
according to the University of Michigan’s Committee on Use and Care of
VWF plasma protein quantitation and analysis
Platelet-poor plasma was isolated from whole blood by centrifugation at
2000g and stored at ?70°C before analysis. VWF levels were quantitated
by enzyme-linked immunosorbent assay (ELISA) essentially as previously
described.17A pool of male and female adult B6 plasma (age, 6-8 weeks)
was used to generate a standard curve, and a mean VWF level was
calculated for each mouse from the 3 retro-orbital bleeds. Because of age
and sex differences between the standard pool and experimental groups, the
B6 parental strain levels are slightly higher than the value of 10 that was
arbitrarily assigned to the standard.Analysis of variance was performed on
natural log-transformed VWF values to assess the ELISA assay variance
(variation in replicate measurements on the same plasma sample), environ-
mental variance (variation among replicate measurements taken on the
same animal), genetic variance (variation among animals), and total
backcross population variance. Heritability was estimated by comparing
genetic variance with total backcross population variance after correcting
for assay variance.Assay variance was determined to be approximately 2%
of total variance.
Genomic DNA was isolated from liver by digestion in buffer containing
0.1 mg/mL proteinase K, 0.1M Tris (pH 8.0), 0.2M NaCl, 0.2% SDS, and
5mM EDTA at 55°C overnight, followed by phenol/chloroform extraction,
isopropanol precipitation, washing with 70% ethanol, and resuspension in
10mM Tris-Cl, pH 8.0. Genotyping on DNAfrom 200 mice was performed
by the Mammalian Genotyping Service of the National Heart, Lung, and
Blood Institute. Seven mice with VWF levels in the middle of the
population were removed from the 207 N2 progeny, and 157 simple
sequence length polymorphisms (SSLPs) were examined across the ge-
nome. At the University of Michigan standard genotyping was performed
by polymerase chain reaction (PCR) and agarose gel electrophoresis as
described17to type an additional 28 SSLPs and 4 single nucleotide
polymorphisms (SNPs) on all 207 N2 mice, 33 SSLPs selectively applied to
mice with the 5% highest and lowestVWF levels, and 7 SSLPs on the entire
N2 population excluding the 5% highest and lowest VWF groups. All map
positions are noted in millions of base pairs (Mbp), obtained from NCBI
(National Center for Biotechnology Information) Build 37 of the mouse
genome, unless otherwise noted.
Genome scan analysis and QTL identification
A genome scan was performed by standard interval mapping21with the
natural log of the mean VWF level (averaged across the 3 plasma samples)
as a quantitative trait and sex as an additive covariate. LOD (logarithm of
odds) scores were obtained as the log-10 likelihood ratio comparing a
model with a single QTL(quantitative trait locus) to a model with no QTLs.
Because the effect size of the QTL on chromosome 6 was so strong,
2 additional genome scan analyses were also performed with the marker
D6Mit366 included as either an additive or interactive covariate, keeping
the sex adjustment in both cases. The choice of D6Mit366 was arbitrary
because D6Mit329 and D6Mit366 are both at the peak of the chromosome 6
QTL and only 1 Mbp apart. Significance thresholds were obtained by
permutation testing, whereby the genome scan analysis was repeated
50 000 times on the autosomes and 1 000 000 times on the X chromo-
some,22randomly shuffling phenotypes while keeping genotypes fixed.23
The LOD scores for genomewide significance at levels ? equal 0.01 (highly
significant), 0.05 (significant), and 0.25 (suggestive) were taken as quan-
tiles of the corresponding permutation distributions, partitioning the type I
error rate across the autosomes and the X chromosome, according to the
method of Broman et al.22Note that sample sizes in analyses of the
X chromosome were reduced from 207 to 78 individuals, because analysis
was restricted to individuals from crosses for which the X chromosome was
segregating.22Finally, 95% confidence intervals were obtained as 96.5%
Bayes credible intervals.24,25Data management and genome scan analyses
were performed with the use of R26and the R/qtl package.27
Allele-specific primer extension analysis
Three male and 3 female 9-week-old (B6 ? WSB) F1 mice were anesthe-
tized with isoflurane and pentobarbital and humanely killed. Lungs were
dissected and immediately frozen in liquid nitrogen, and total mRNA was
isolated with Trizol (Invitrogen). mRNA was treated with DNAse I
(Invitrogen) and subjected to reverse transcription (RT)–PCR with Super-
Script One-Step (Invitrogen) with the use of Vwf internal exon 7 primers
5?-GGGAGCAATGCCAGCTACT-3? and 5?-GGCACTGTGGTCAGTC-
CAG-3?, at 50°C for 30 minutes, 94°C for 2 minutes, 35 cycles of 94°C for
30 seconds, 56°C for 30 seconds, 72°C for 1 minute, concluding with 72°C
for 10 minutes. Relative allele-specific mRNA accumulation was deter-
mined using a primer extension assay with fluorescently labeled primers as
previously described,17,28using a standard curve composed of B6 and WSB
genomic DNA quantitated with PicoGreen (Invitrogen) and mixed in
various ratios. The RT-PCR product was treated with ExoSAP-IT (US
Biochemical Corp) to remove free nucleotides and primers. Primer
extension was carried out with Thermo Sequenase (US Biochemical Corp),
a 6-FAM–labeled primer (5?-TGGATCCCGAGTCCTTTGTGGCTC-3?)
and a mixture of nucleotides containing ddCTP and run at 94°C for
2 minutes, 30 cycles of 94°C for 30 seconds, 65°C for 30 seconds, 72°C for
1 minute, resulting in differentially sized products because of a T/C SNP
?758 bp from the A of the initiation methionine of Vwf. Products were
diluted 1:13 in Hi-Di formamide (Applied Biosystems), separated on an
Applied Biosystems 3730XL DNAAnalyzer at the University of Michigan
Sequencing Core, and quantitated with GeneMarker 1.51 (SoftGenetics
LLC). The standards were fit to a line resulting in an R2value of 0.996,
calculated by linear regression with Microsoft Excel 2003 (Microsoft
Calculation of Vwf locus contribution to plasma VWF levels in
the N2 population
The following calculation was used to assess the relative contribution of the
Vwf locus to the level of plasma VWF. The N2 population was subdivided
into 2 groups based on genotype at a G/T SNPin exon 5 of Vwf (?365 base
pairs from the A of the initiation methionine) that produces an XhoI
restriction fragment polymorphism that cuts WSB, but not B6 (amplified
with the primers 5?-GGCAAGAGAATGAGCCTGTC-3? and 5?-TGAAT-
CACAGAATCAATGGACTA-3?), and the average VWF level was calcu-
lated for the resulting B6:B6 and B6:WSB groups.Any minor modifier loci
would be expected to be evenly divided among these 2 groups by
Mendelian inheritance; therefore, their effects on plasma VWF levels
should be evenly distributed.
VWF plasma levels in WSB/EiJ mice are 3.5-fold higher than
VWF levels were quantitated by ELISA on plasma obtained from
6 laboratory inbred strains as well as the wild-derived inbred strain
WSB. VWF plasma protein levels were determined as the average
of data from 3 separate retro-orbital bleeds, done at least 1 week
apart.WSB exhibited the highest plasmaVWF, with levels 3.5-fold
greater than most of the common inbred strains, including B6
(Figure 1). Two 129/Sv substrains tested each displayed intermedi-
ate levels of VWF, which were 2-fold greater than B6.
VWF MODIFIERS IN INBRED STRAINS OF MICE5369BLOOD, 17 DECEMBER 2009?VOLUME 114, NUMBER 26
(B6 ? WSB) N2 progeny display a wide range of VWF levels
B6 was chosen to cross with WSB because it has been used as a
standard strain in many previous hemostasis and other disease
models, and it is also the reference strain for the mouse ge-
nome.29,30Importantly, neither B6 nor WSB contain allelic variants
associated with the previously reported modifier loci Mvwf1 and
Mwf2.17,31(B6 ? WSB) F1 plasma VWF levels were intermediate
between the 2 parental strains but closer to WSB (Figure 2).
Therefore, an outcross/backcross approach with B6 as the back-
cross parent was chosen. Plasma VWF levels were determined on
207 N2 progeny generated from a (B6 ? WSB) F1 backcross to B6
(Figure 2). The observed levels in N2 animals encompassed the
entire phenotypic range, extending to the pure parental strains and
peaking just below the level of F1 mice. Heritability ofVWF levels
in the (B6 ? WSB) N2 population was determined to be approxi-
mately 71%. There was also a statistically significant difference in
population averages among bleeds (population averages: bleed 1,
24.5; bleed 2, 18.2; bleed 3, 16.4; P ? .001).
Genotyping of N2 backcross progeny identifies 3 major QTLs
for VWF plasma levels
Two hundredN2progenyweregenotypedwith189 markersspaced
across the genome. Additional markers were used in several sub-
sets of mice (see “Genotyping”). Standard interval mapping at
1-centimorgan (cM) intervals using the R/qtl package27was
performed on the natural log-transformed mean VWF levels for the
N2 population with adjustment for sex.Three QTLs were identified
with significant linkage to VWF levels, with peak markers at
D6Mit329 (Mvwf5), D10Mit269 (Mvwf6), and D5Mit66 (Mvwf7)
and LOD scores of 11.65, 3.72, and 3.22, respectively (Figure 3A;
To detect additional minor QTLs masked by the extremely
strong effect of the chromosome 6 locus, the data were reanalyzed
with the marker D6Mit366 (115.2 Mbp) as an additive or interac-
tive covariate. The results were very similar to the initial analysis,
without significant changes in the statistical significance of the
LOD scores for Mvwf6 and Mvwf7 (Figure 3B-C). No additional
loci, including the known modifier loci B4galnt2 (Mvwf1), Mvwf3-
4,32and the humanABO blood group ortholog, Abo (chromosomes
11, 4, 13, and 2, respectively), surpassed the 0.05 significance
threshold in any of these analyses (as determined by permuta-
Examination of candidate genes in the peak of the Mvwf5 QTL
showedthattheVwfgeneitselfislocatedinthisregion.(B6 ? WSB)
N2 mice that were BW (B6:WSB) at both the D6Mit329 and
D6Mit339 markers (114.0 and 136.3 Mbp, respectively, flanking
the Vwf locus) were backcrossed to B6 to the N4 and N5
generations, with selection for heterozygosity at both markers.
VWF levels were measured as described above on mice that were
BB (B6:B6, non-BW littermates derived during backcrossing) or
BW at both markers (Figure 4 inset). There was a statistically
significant increase in VWF levels in the BW mice (19.6 vs 14.6 in
BB mice), confirming that Mvwf5 is a true QTL and narrowing the
candidate region to 11.6 cM, which includes Vwf (Figure 4).
Strain-specific differences at the Vwf locus alter steady state
Vwf mRNA levels
Sequencing of the WSB Vwf cDNA followed by comparison to
published B6 sequence revealed 9 nonsynonymous SNPs, all
localized to the VWF propeptide (Table 2). Eighteen synonymous
SNPs were discovered, with a large block devoid of any variation at
the 3? end (Table 2). This represents a greater level of divergence
between B6 and WSB, 2 strains that are thought to be primarily
mus musculus domesticus in origin, versusA/J and CASA/RkJ (the
strains used to identify Mvwf2-4), which are mus musculus
domesticus and mus musculus castaneus, respectively.17
To evaluate potential cis-regulatory differences in mRNA
expressionorstability,lungmRNAwasisolatedfrom(B6 ? WSB)
F1 mice and subjected to RT-PCR for Vwf mRNA, followed by
primer extension analysis that distinguishes the B6 and WSB
alleles because of an exonic SNP (Figure 5A). A similar approach
has been successful in distinguishing expression from maternal and
paternal alleles in cases of human VWD.33Quantitation showed
that 38.7% (? 0.6%) of transcripts were derived from the B6 allele
and 61.3% from the WSB locus (Figure 5B).
Subdivision of the N2 population into 2 groups based on
genotype at Vwf (see “Methods”) resulted in an average VWF level
of 18.5 in the B6:B6 group and 22.7 in the B6:WSB group. The
B6:B6 group represents the contribution of 2 B6 alleles of Vwf (or
9.25 per B6 allele), whereas B6:WSB represents 1 B6 and 1 WSB
allele (or 22.7 ? 9.25 ? 13.45 for WSB). Thus, the B6 allele
contributes 41% of plasma VWF in B6:WSB heterozygotes
(9.25/22.7), versus 59% for the WSB allele (13.45/22.7). The
results of these calculations are indistinguishable from the mRNA
data determined by primer extension (38.7% B6 and 61.3% WSB;
P ? .6 by ?2analysis with the plasma values as the expected
values). Therefore, we conclude that the Vwf locus contribution to
VWF plasma protein levels in the B6xWSB N2 population can be
fully explained by differential mRNA accumulation from the
2 alleles. Additional minor contributions from the Vwf gene to the
VWF antigen (relative to B6 pool)
Figure 1. Inbred mouse strain survey for VWF plasma protein levels. Individual
mice were bled, and the results were averaged and normalized to an adult B6 plasma
pool arbitrarily assigned a value of 10. Error bars represent SD.
VWF antigen (relative to B6 pool)
Number of mice
10 13 16 19 22 25 28 31 34
Figure 2. Histogram of N2 generation VWF plasma levels. Individual N2 mice
were bled, and the results were averaged and normalized to an adult B6 plasma pool
that was arbitrarily assigned a value of 10. The VWF antigen levels were binned and
plotted as indicated on the x-axis. The locations of the parental strain and
(B6 ? WSB) F1 hybrid values are indicated by ™. These are 13.1, 24.8, and 35.3, for
B6, F1, and WSB, respectively.
5370 SHAVIT et alBLOOD, 17 DECEMBER 2009?VOLUME 114, NUMBER 26
difference in plasma VWF levels between these strains, such as
biosynthesis or clearance resulting from the nonsynonymous SNPs,
cannot be completely excluded but are probably small.
Mvwf6 overlaps with human VWF QTL
The 96.5% Bayes credible intervals were constructed for Mvwf6
and Mvwf7 (Figure 6), as previously described.25Human ortholo-
gous regions were determined from comparative orthology maps34
and aligned to potential VWF modifier regions identified in human
populations. Souto et al (from the Genetic Analysis of Idiopathic
Thrombophilia or GAIT study)35analyzed VWF levels as a
quantitative trait in a group of 21 Spanish families and found
potential linkage to several regions unlinked to the human VWF
locus. The linkage identified in the GAIT study included 22q11.1,
which displays orthology to Mvwf6 (Figure 6). Similar compari-
sons to linkage data from a large Amish pedigree phenotyped by
ristocetin cofactor activity36reveal orthology for Mvwf6 with
human loci at 12q12 and 21q22.3 (Figure 6).
Previous analyses of laboratory strains of mice have identified both
major and minor loci regulating VWF levels.17,18,32We now report a
second strong Vwf variant associated with regulation of VWF plasma
protein levels, as well as 2additional minor loci that are unlinked to the
Vwf locus. Using 2previously unexamined strains, we found the
found in an (A/J?CASA/RkJ) F2 population17and also similar to
estimates of VWF level heritability in human populations.9,10The
(B6?WSB) N2 population displayed a statistically significant de-
to adulthood (5-8.5weeks). This may parallel the pattern in humans, in
which plasma VWF at birth decreases by 43% to near adult levels by
6months of age.37In contrast, the (A/J?CASA/RkJ) F2 population17
did not show a similar age-dependent decrease in VWF. These data
show strain-specific differences in the regulation of VWF levels over
Figure 3. R/qtl analysis of N2 progeny shows 3 QTLs that modify VWF plasma levels. Interval mapping was performed on natural log-transformed mean VWF levels.
(A) LOD scores with adjustment for sex show 3 QTLs on chromosomes 6, 10, and 5 (Mvwf5, Mvwf6, and Mvwf7, respectively). No additional loci were identified by analysis for
additive (B) or epistatic (C) effects upon adjustment for Mvwf5. Solid, dashed, and dotted lines indicate ? ? 0.25 (suggestive), 0.05 (significant), and 0.01 (highly significant)
thresholds, respectively, obtained by permutation testing.
Table 1. QTLs identified with significant linkage to plasma VWF
Locus Chromosome Position, MbpPeak marker LODP
Standard interval mapping was performed on natural log-transformed mean VWF levels from the B6 ? WSB N2 population at 1-cM intervals by using the R/qtl package
with adjustment for sex. P values represent the genomewide significance of LOD scores as determined by permutation testing.
QTLindicates quantitative trait locus; VWF, von Willebrand factor; and LOD, logarithm of odds.
VWF MODIFIERS IN INBRED STRAINS OF MICE5371 BLOOD, 17 DECEMBER 2009?VOLUME 114, NUMBER 26
Mvwf5 is a natural mouse allele altering plasma VWF because
of a cis-regulatory mutation in the Vwf gene itself. This is the
second example of a natural variant Vwf allele among inbred strains
of mice, as Mvwf2 is due to a VWF gene-coding mutation.17The
relative elevation in steady-state levels of Vwf mRNAderived from
the WSB Mvwf5 allele could be due to an alteration in
transcriptional regulation, splicing, or mRNA stability, mecha-
nisms identified in many common human genetic disorders, such as
Although most human type 1 VWD mutations identified to date
affect protein function, transport, secretion, or clearance, this
probably represents an ascertainment bias because most studies
only examine exons, exon/intron junctions, and the proximal
Consistent with this hypothesis, comprehensive exon sequencing in
patients with type 1 VWD has identified mutations in only 67% of
persons.12,16,39,40Of those with identified mutations, 7% altered
splice sites and 11% were localized to the promoter. Although not
tested directly, both classes of mutation would be expected to alter
allelic expression at the mRNA level, as shown here for murine
Mvwf5. In addition, a subset of the larger class of exonic mutations
could also potentially affect mRNA levels through alterations in
mRNA stability. Distant regulatory mutations not covered in the
human sequence analysis performed to date could also affect
mRNAexpression, potentially accounting for a significant percent-
age of the above patients for whom no mutations were identified.
Recent data suggest that long-range effects of distant regulatory
elements could be particularly important for VWF gene expres-
sion.41-43Thus, a significant contribution from the large quantity of
VWF intergenic and intronic sequences remains to be explored.
The presence of SNPs within the murine Vwf mRNA
sequence permitted the dissection of relative mRNA expression
from each Vwf allele in this and a previous study.17An analogous
approach has been used for peripheral blood platelet VWF
mRNA in humans.33Future studies applying similar RNA SNP
analysis to human patients could potentially define novel
subgroups of type 1 VWD.
Our findings, taken together with previous work,17,18,32
provide genetic analysis of 5 mouse strains in 3 different crosses
and has identified 2 independent, naturally occurring Vwf gene
mutations leading to altered plasma VWF levels. These data
suggest that the equivalent of type 1 VWD is remarkably
common in mice, as well as in humans. Reports of VWD in
horse, cat, pig, rabbit, and dog5,44suggest that a high prevalence
B6 Mvwf5 BBMvwf5 BW
D6Mit329 Vwf D6Mit339
BB 3.5 8.1
VWF antigen (relative to B6 pool)
Figure 4. VWF levels in congenic mice. VWF plasma
levels were determined. B6 represents C57BL/6J mice
purchased from The Jackson Laboratory (n ? 7). Mvwf5
BW mice were backcrossed to B6 to the N4 or N5
generation, and VWF levels on littermates that were BB
(n ? 18) or BW (n ? 11) at both D6Mit329 and D6Mit339
were determined (see inset; numbers indicate centimor-
gan [cM] distances between markers and Vwf indicates
the XhoI restriction fragment polymorphism described in
“Methods”). *The difference between these 2 groups is
statistically significant (P ? .001 by t test). **The differ-
ence between these 2 groups is statistically significant
(P ? .004 by t test). There was no significant difference
between the B6 and BB groups (P ? .1 by t test). Error
bars represent SD.
Table 2. Location of SNPs in coding sequence of mouse Vwf
Position VWF domainB6 WSBAmino acid ?
mRNA was isolated from WSB lung and reverse transcribed, and polymerase
chain reaction was performed to isolate fragments of Vwf for sequencing. B6
sequence was obtained from NCBI Build 37 of the mouse genome. VWF domains
were deduced by comparison to the human amino acid sequence.
— indicates there is no amino acid change caused by a SNP.
5372SHAVIT et alBLOOD, 17 DECEMBER 2009?VOLUME 114, NUMBER 26
of Vwf gene mutations may be common among all mammalian
species. These observations, together with the highly variable
levels of plasma VWF in humans45and mice (Lemmerhirt et al17
and Johnsen et al31; Figure 1), might be explained by common
selective pressures, potentially including interactions with infec-
tious pathogens. Consistent with this hypothesis, analysis of
B4galnt2 (Mvwf1) in wild mouse populations suggests that this
locus is under selective pressure.46These observations also
suggest that the genetic regulation of plasma VWF in the inbred
laboratory mouse is complex and probably involves a large
number of genes. The corresponding picture in the outbred
human population is probably more complex and even more
difficult to approach experimentally.
The orthologous region of Mvwf6 overlaps with a significant
VWF QTL identified in the GAIT study,35as well as regions
identified as potential human VWD modifiers in a large Amish
pedigree.36These human loci are distinct from those showing
potential conservation of synteny with Mvwf3 and Mvwf4.32Thus,
analyses of inbred mouse strains have identified 5 potential modi-
fier loci outside of the Vwf gene, Mvwf1,18Mvwf3-4,32and Mvwf6-7
(this report), 3 of which display conservation of synteny with
potential human modifier loci. Although the overlapping regions
are still quite large, positional cloning of theseVWF modifier genes
in the mouse should identify candidate genes for the modification
of bleeding and thrombotic risk in humans.
We thank the National Heart, Lung, and Blood Institute Mamma-
lian Genotyping Service; the University of Michigan Sequencing
and Genotyping Core; and Dave Siemieniak, Tricia Hayes, Diana
Keung, Jennifer Myaeng, Susan Spaulding, Beverly Twiss, and
Alok Swaroop for technical assistance.
This work was supported by the National Institutes of Health
(R37-HL39693 and P01-HL057346, D.G.; and R01-GM074244,
K.W.B.), the American Heart Association (0675025N, J.A.S.), the
National Hemophilia Foundation Clinical Fellowship Program
(J.A.S.), the National Institute of Child Health and Human
tion Graduate Fellowship Awards (H.L.L. and A.M.). D.G. is an
investigator of the Howard Hughes Medical Institute.
%B6 (gDNA diluted in WSB gDNA)
%B6/(B6+WSB) (primer extension product)
Figure 5.Allele-specific expression analysis of Vwf in (B6 ? WSB) F1 mice.Adult lung cDNAwas prepared from F1 mice (n ? 3 males and 3 females). Polymerase chain
reaction was performed with exonic primers flanking a T/C SNP, followed by primer extension with a fluorescently labeled primer and dATP, dGTP, dTTP, and ddCTP, which
resultsindifferentiallysizedproductsineachstrain (A).Fluorescentprimerextensionproductswereseparated,quantitated,andcomparedwith genomicstandards(B).B6and
WSB genomic DNA were mixed in various proportions to produce the standard curve. Results are expressed as a percentage of B6 transcripts from total transcripts. Linear
regression was performed with Microsoft Excel.
Figure 6. Mvwf QTL 96.5% Bayes credible intervals. Chromosomes are repre-
sented as vertical bars, and ticks represent markers used in mapping. To the left of
each chromosome is the 96.5% Bayes credible interval constructed as described.25
To the right of chromosome 10 are the regions with human homology of synteny to
significant regions identified in human studies 22q11.1,3521q22.3, and 12q12.36
JS1415 is a marker derived from a B6/WSB T/G SNP downstream from the Vwf gene
(128.3 Mbp) that produces an MspI restriction fragment polymorphism.
VWF MODIFIERS IN INBRED STRAINS OF MICE 5373BLOOD, 17 DECEMBER 2009?VOLUME 114, NUMBER 26
Contribution: J.A.S. participated in research design, performed
the research, and wrote the paper; A.M. aided in data analysis
and wrote the paper; H.L.L. participated in research design and
performed the research; K.W.B. aided in data analysis and wrote
the paper; and D.G. participated in research design and wrote
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
The current affiliation for A.M. is Department of Biomedical
Engineering, University of Virginia, Charlottesville, VA. The
current affiliation for K.W.B. is Department of Biostatistics and
Medical Informatics, University of Wisconsin, Madison, WI.
Correspondence: David Ginsburg, Howard Hughes Medical Insti-
tute, University of Michigan, 210Washtenaw Ave, Life Sciences
1. Vischer UM. von Willebrand factor, endothelial
dysfunction, and cardiovascular disease.
J Thromb Haemost. 2006;4(6):1186-1193.
2. KosterT,BlannAD,Brie ¨tE,VandenbrouckeJP,
3. Kraaijenhagen RA,Anker PSI, Koopman MMW,
et al. High plasma concentration of factor VIII is a
major risk factor for venous thromboembolism.
Thromb Haemost. 2000;83(1):5-9.
4. RosendaalFR.Highlevelsoffactor VIIIandvenous
5. Levy GG, Ginsburg D. Getting at the variable ex-
pressivity of von Willebrand disease. Thromb
6. Sadler JE. Von Willebrand disease type 1: a diag-
nosis in search of a disease. Blood. 2003;101(6):
7. Vossen CY, Hasstedt SJ, Rosendaal FR, et al.
Heritability of plasma concentrations of clotting
factors and measures of a prethrombotic state in
a protein C-deficient family. J Thromb Haemost.
8. Souto JC,Almasy L, Borrell M, et al. Genetic de-
terminants of hemostasis phenotypes in Spanish
families. Circulation. 2000;101(13):1546-1551.
9. de Lange M, Snieder H,Ariens RA, Spector TD,
Grant PJ. The genetics of haemostasis: a twin
study. Lancet. 2001;357(9250):101-105.
10. Orstavik KH, Magnus P, Reisner H, Berg K,
Graham JB, Nance W. Factor VIII and factor IX in
a twin population. Evidence for a major effect of
ABO locus on factor VIII level. Am J Hum Genet.
11. Gill JC, Endres-Brooks J, Bauer PJ, Marks WJ,
Montgomery RR. The effect ofABO blood group
on the diagnosis of von Willebrand Disease.
13. Eikenboom J, Van Marion V, Putter H, et al. Link-
age analysis in families diagnosed with type 1
von Willebrand disease in the European study,
molecular and clinical markers for the diagnosis
and management of type 1 VWD. J Thromb
14. James PD, PatersonAD, Notley C, et al. Genetic
linkage and association analysis in type 1 von
Willebrand disease: results from the Canadian
type 1 VWD study. J Thromb Haemost.
15. Lanke E, JohanssonAM, Hallden C, Lethagen S.
Genetic analysis of 31 Swedish type 1 von Wille-
brand disease families reveals incomplete link-
age to the von Willebrand factor gene and a high
frequency of a certain disease haplotype.
J Thromb Haemost. 2005;3(12):2656-2663.
16. GoodeveA. Genetics of type 1 von Willebrand
disease. Curr Opin Hematol. 2007;14(5):444-449.
17. Lemmerhirt HL, Shavit JA, Levy GG, Cole SM,
Long JC, Ginsburg D. Enhanced VWF biosynthe-
sis and elevated plasma VWF due to a natural
variant in the murine Vwf gene. Blood. 2006;
18. Mohlke KL, PurkayasthaAA, Westrick RJ, et al.
Mvwf, a dominant modifier of murine von
Willebrand factor, results from altered lineage-
specific expression of a glycosyltransferase. Cell.
19. Haberichter SL, Castaman G, Budde U, et al.
Identification of type 1 von Willebrand disease
patients with reduced von Willebrand factor sur-
vival by assay of the VWF propeptide in the Euro-
pean study: molecular and clinical markers for the
diagnosis and management of type 1 VWD
(MCMDM-1VWD). Blood. 2008;111(10):4979-
20. Millar CM, RiddellAF, Brown SA, et al. Survival of
von Willebrand factor released following DDAVP
in a type 1 von Willebrand disease cohort: influ-
ence of glycosylation, proteolysis and gene muta-
tions. Thromb Haemost. 2008;99(5):916-924.
21. Lander ES, Botstein D. Mapping mendelian fac-
tors underlying quantitative traits using RFLP link-
age maps. Genetics. 1989;121(1):185-199.
22. Broman KW, Sen S, Owens SE, ManichaikulA,
Southard-Smith EM, Churchill GA. The X chro-
mosome in quantitative trait locus mapping. Ge-
23. Churchill GA, Doerge RW. Empirical threshold
values for quantitative trait mapping. Genetics.
24. Sen S, Churchill GA.Astatistical framework for
quantitative trait mapping. Genetics. 2001;159(1):
25. ManichaikulA, Dupuis J, Sen S, Broman KW.
Poor performance of bootstrap confidence inter-
vals for the location of a quantitative trait locus.
26. Ihaka R, Gentleman R. R: a language for data
analysis and graphics. J Comput Graph Stat.
27. Broman KW, Wu H, Sen S, Churchill GA. R/qtl:
QTL mapping in experimental crosses. Bioinfor-
29. Battey J, Jordan E, Cox D, Dove W.An action
plan for mouse genomics. Nat Genet. 1999;21(1):
30. Waterston RH, Lindblad-Toh K, Birney E, et al.
Initial sequencing and comparative analysis of
the mouse genome. Nature. 2002;420(6915):
31. Johnsen JM, Levy GG, Westrick RJ, Tucker PK,
Ginsburg D. The endothelial-specific regulatory
mutation, Mvwf1, is a common mouse founder
allele. Mamm Genome. 2008;19(1):32-40.
32. Lemmerhirt HL, Broman KW, Shavit JA, Ginsburg
D. Genetic regulation of plasma von Willebrand
factor levels: quantitative trait loci analysis in a
mouse model. J Thromb Haemost. 2007;5(2):
ProcNatlAcadSciU S A.1991;88(9):3857-3861.
34. The Jackson Laboratory. Mammalian Orthology.
Accessed January 29, 2009.
35. Souto JC,Almasy L, Soria JM, et al. Genome-
wide linkage analysis of von Willebrand factor
plasma levels: results from the GAIT project.
Thromb Haemost. 2003;89(3):468-474.
37. Andrew M, Paes B, Milner R, et al. Development
of the human coagulation system in the full-term
infant. Blood. 1987;70(1):165-172.
38. Cunningham MJ, Sankaran VG, Nathan DG,
Orkin SH. The thalassemias. In: Orkin SH,
Nathan DG, Ginsburg D, LookAT, Fisher DE, Lux
SE, eds. Nathan and Oski’s Hematology of In-
fancy and Childhood. 7th ed. Philadelphia, PA:
39. GoodeveA, Eikenboom J, Castaman G, et al.
Phenotype and genotype of a cohort of families
historically diagnosed with type 1 von Willebrand
disease in the European study, Molecular and
Clinical Markers for the Diagnosis and Manage-
ment of Type 1 von Willebrand Disease
(MCMDM-1VWD). Blood. 2007;109(1):112-121.
40. James PD, Notley C, Hegadorn C, et al. The mu-
tational spectrum of type 1 von Willebrand dis-
ease: results from a Canadian cohort study.
41. Bernat JA, Crawford GE, OgurtsovAY, Collins
FS, Ginsburg D, KondrashovAS. Distant con-
served sequences flanking endothelial-specific
promoters contain tissue-specific DNase-
hypersensitive sites and over-represented motifs.
Hum Mol Genet. 2006;15(13):2098-2105.
42. KleinschmidtAM, Nassiri M, Stitt MS, et al. Se-
quences in intron 51 of the von Willebrand factor
gene target promoter activation to a subset of
lung endothelial cells in transgenic mice. J Biol
43. Hough C, Cameron CL, Notley CR, et al. Influ-
ence of a GT repeat element on shear stress re-
sponsiveness of the VWF gene promoter.
J Thromb Haemost. 2008;6(7):1183-1190.
44. Ginsburg D, Bowie EJW. Molecular genetics of
von Willebrand disease. Blood. 1992;79(10):
45. Nichols WC, Ginsburg D. von Willebrand disease.
Medicine (Baltimore). 1997;76(1):1-20.
46. Johnsen JM, Teschke M, Pavlidis P, et al. Selec-
tion on cis-regulatory variation at B4galnt2 and its
influence on von Willebrand factor in house mice.
Mol Biol Evol. 2009;26(3):567-578.
5374SHAVIT et alBLOOD, 17 DECEMBER 2009?VOLUME 114, NUMBER 26